This invention relates generally a fluorescence microscopy technique to three dimensional (3D) imaging of live biological specimens in real time.
Cells live in 3D environments. A more accurate understanding of cell behaviors and cell-cell interactions can be obtained by studying cells in their native, multi-cellular environments than when cultured on substrates. In a multi-cellular organism, the intracellular activities of a cell affect not only the cell itself but also its neighbor cells, and the cellular behavior of a cell results from both its own intracellular activities and its interactions with other cells. In other words, cell behaviors in a multi-cellular process are caused by the intracellular activities of all cells involved in the process. Thus, in order to understand cell behaviors in a multi-cellular process, it is necessary to study all involved cells at sub-cellular level to acquire the dynamic information of their intracellular activities, by which the underlying connections between cellular behaviors and intracellular activities of the involved cells can be revealed. For this reason, fluorescence imaging techniques that allow low-invasive 3D imaging of multi-cellular specimens with high spatial and temporal resolution are required.
Multi-cellular specimens are difficult to image in 3D because of their size and complexity accompanied with optical aberrations and light scattering. Both high 3D spatial resolution and good optical sectioning capability are required in a large field of view (FOV) in order to visualize the specimen with sub-cellular, or even cellular structural details. Meanwhile, the photobleaching and photodamage must be low enough to allow live imaging at needed speed for a certain period of time. Therefore, selective plane illumination microscopy (SPIM) is getting increasing attention for its advanced 3D live imaging ability [1-4]. Generally, the latest SPIM techniques allow low-invasive, high-speed 3D imaging of either single cell specimens with sub-cellular level, submicron 3D spatial resolution [5-8], or multi-cellular specimens with cellular level spatial resolution of a few microns [9-12], although the actual performance varies depending on the sample and fluorophore. However, imaging multi-cellular specimens in 3D with sub-cellular spatial resolution remains challenging despite the progress that has been made in SPIM development.
SPIM obtains 3D imaging ability by confining the illumination light near the detection focal plane with a light sheet. With a given detection numerical aperture (NA), its 3D imaging ability, including the spatial resolution, optical sectioning capability, field of view (FOV), photobleaching and photodamage, is mainly determined by the intensity profile of the light sheet [13, 14]. A uniformly thin and large light sheet is therefore required in SPIM to maximize its imaging ability, and the generation of such a light sheet has been a major focus of SPIM development [5-7, 15, 16]. Although different methods have been developed, none of them are ideal. Essentially, every light sheet balances the properties of light sheet thickness, the illumination light confinement, and the light sheet size differently, which results in different 3D spatial resolution, optical sectioning capability and FOV, respectively [13, 14]. Nevertheless, it becomes extremely difficult to balance these properties as the FOV increases to image multi-cellular specimens of dozens of microns or larger, since an ideal light sheet that has thin thickness, good light confinement and a large size at the same time does not exist due to the diffraction of light. Either the spatial resolution, optical sectioning capability, or both must be sacrificed to reach a larger FOV because the light sheet either becomes thicker, or the excitation light is less confined as its size increases. Therefore, the tradeoff between the spatial resolution, optical sectioning capability and FOV sets a fundamental limit on conventional SPIM, and a key problem of imaging multi-cellular specimens with sub-cellular spatial resolution using SPIM turns out to be how to increase the FOV without losing the spatial resolution and optical sectioning capability. Different approaches other than finding a perfect light sheet must be made.
Multiview SPIM is a different approach that works in two ways to improve the 3D imaging ability of SPIM on multi-cellular specimens. First, by sending the excitation light sheet and collecting the fluorescence signal from different directions, the final image is less affected by the optical aberration and light scattering introduced by the sample [9-12]. Next, 3D images taken from different directions can be fused together with similar methods used in tomography, by which the 3D spatial resolution can exceed the resolution limit set up by the light sheet in theory [1, 8, 12]. Nevertheless, the improvement in resolution by this approach relies on the number of different view directions, and the spatial resolution and the signal to noise ratio (SNR) of the 3D images taken in these different views. In multiview SPIM, the number of view directions is limited to two (lateral and axial) without rotating the sample, while the spatial resolution and SNR of the 3D image taken in each view are still strictly constrained by the light sheet used to take the image. Therefore, multiview SPIM cannot bypass the fundamental tradeoff of SPIM set up by the light sheet. Furthermore, high-speed sub-cellular dynamics, the optical aberration and light scattering introduced by both the sample and the agarose gel usually used to mount the sample make it more difficult to fuse different views accurately with high spatial resolution. Consequently, multiview SPIM is suited for imaging relatively large and bright multi-cellular specimen with cellular level spatial resolution. Imaging multi-cellular specimens with sub-cellular spatial resolution remains a problem with multiview SPIM.
The nonexistence of a perfect light sheet not only limits the 3D imaging ability of SPIM, but also creates a severe yet underappreciated practical problem. Optimization of the light sheet, including its type and dimensions, is required case by case in SPIM depending on the sample to be imaged and the biological question to be studied, as different light sheets balance the 3D imaging ability of SPIM differently [14]. The light sheet is usually determined based on the prior knowledge of the specimen and the biological process to be imaged, and it remains the same during the entire imaging process because a realignment of the microscope, that often takes hours even for SPIM experts, is usually required to change the light sheet. It is not only extremely inconvenient, but also prevents SPIM from adapting itself to reach the optimal imaging performance, because different organelles have different structures and dynamics, and both of these can change in live imaging. Instead, the optimization of SPIM imaging performance would become a closed-loop process if the light sheet could be adjusted in real-time using the immediate imaging result as feedback. More importantly, the understanding of a biological process relies on the spatial and temporal information that can be obtained from the process, whereas an ideal method that is capable of acquiring both with a high resolution is often unavailable, especially for multi-cellular specimens. The only option under such circumstances is to compromise either the spatial or the temporal resolution strategically, and only acquire the necessary information needed to understand a biological process. Such ability and flexibility to compromise can only be obtained by adjusting the light sheet in real-time in SPIM. Thus, be able to optimize the excitation light sheet in real-time is another key to improving the 3D live imaging ability of SPIM on multi-cellular specimens.